Epitope tagging is a technique in which a known epitope tag (typically 6 to 30 amino acids) is fused to a recombinant protein by placing sequence encoding the epitope within the same open reading frame of the protein by means of genetic engineering. By choosing an epitope tag for which an antibody is available, the technique makes it possible to detect tagged proteins for which no antibody is available. By selection of the appropriate epitope tag and antibody pair, it is possible to find a combination with properties that are suitable for the desired experimental application, such as Western blot analysis, immunoprecipitation, immunochemistry, and affinity purification, amongst others.
The first commercially available tags were originally designed for protein purification. Examples of these early commercial products include FLAG, 6×His, and glutathione-S-transferase (GST) systems. The FLAG tagging system in its original version included the anti-FLAG M1 monoclonal antibody with calcium-dependent binding. FLAG-tagged proteins can be eluted from the M1 antibody with EDTA (Hopp et al. 1988). Likewise, the 6×His tag is used for purification of recombinant proteins by means of metal chelate chromatography (Hochuli et al. 1988). Similarly, GST-tagged proteins can be purified using glutathione agarose (Smith et al. 1988). In addition to the commercial tags, the development of other tags such as HA (Field et al. 1988) and c-myc (Evan et al. 1985) were reported. As research progressed and recombinant DNA technology evolved, the utility of epitope tags in the study of protein interaction was recognized. For example, the anti-FLAG M2 monoclonal antibody was made available commercially (Brizzard et al. 1994) as were monoclonal antibodies for the 6×His tag (Kaufmann et al. 2002), HA tag, and c-myc tag. Today, there are numerous types of tags with different features suited to diverse applications (Jarvik and Telmer 1998).
In addition to the traditional antibody and epitope combinations, other types of epitope tags have been discovered. An early example is the protein A tag, which binds to IgG (Uhlén et al. 1983). Other examples include those based on interaction with streptavidin (Schmidt and Skerra 2007) and biotin (Tucker and Grisshammer 1996), maltose-binding peptide (MBP tag) and maltose (di Guan et al. 1988), chitin-binding domain (CBD) and chitin (Chong et al. 1997), and the calmodulin-binding peptide that binds to calmodulin (Rigaut et al. 1999). Another type of tag is the S-peptide tag that binds to the S-protein derived from pancreatic RNAse A (Hackbarth et al. 2004).
Further advancements in the field are the use of multiple epitope tags to increase signal strength and signal-to-noise ratio, e.g., multiple copies of FLAG, His, c-myc and HA tags, or the use of tandem copies of different tags. For example, in tandem affinity purification (TAP), used for the study of protein networks, an IgG-binding domain and calmodulin-binding peptide with an intervening tobacco etch virus (TEV) protease cleavage site are used (Rigaut et al. 1999). Recently, a shorter TAP tag has been described, the SF-TAP tag (Gloeckner et al. 2007), in which two tandem copies of the Strep-tag II were combined with the FLAG tag.
While the use of epitope-tagging facilitates the study and characterization of newly discovered proteins, the technique does have limitations. The insertion of (an) epitope tag(s) can alter protein function, especially when a large tag is used (e.g., GST and MBP tag). Notably, the relatively short epitope tags, such as FLAG, rarely affect the properties of the heterologous protein of interest and are very specific for their respective primary antibodies. However, antibody affinity chromatography often involves low or high pH elution, which can irreversibly affect the properties of the fusion protein. It also employs resin, which has limited reusability. Poly-Histidine, typically in the form of a hexa-His tag, appended to the N- or C-terminus of a recombinant polypeptide, has been widely used to purify recombinant proteins using immobilized metal ion chromatography (IMAC). However, only moderate purity from Escherichia coli extracts and relatively poor purification from yeast, Drosophila and HeLa extracts are retrieved using a (His)6 tag (Lichty et al. 2005). Also, aspecific binding of contaminating proteins (e.g., E. coli SlyD, a prolyl isomerase) is often observed in IMAC preparations of heterologously expressed (His)6-tagged proteins, due to histidine clusters or intrinsic metal binding sites on these proteins. Further, few of the tags that have been described in literature have been tested in a high-throughput context. Peptide epitopes like the FLAG-tag, the calmodulin-binding peptide, the Strep-tag or Streptag II and the biotin acceptor peptide all exhibit a high degree of specificity for their cognate binding partners. However, the resins (immobilized proteins) that they interact with tend to be expensive, are easily fouled and have relatively low binding capacities, making them less than ideal for high-throughput applications. Several other advantages and limitations of epitope tagging are listed in Jarvik and Telmer (1998), Kimple and Sondek (2004), Waugh (2005), and Brizzard (2008).
Given that epitope tagging has the potential to make major contributions to the emerging fields of functional genomics and proteomics, there remains a need for alternative epitope tags. Indeed, future applications in these fields will increasingly depend on the separate tagging of individual proteins, requiring multiple epitope tags that can be used in combination.